U.S. patent application number 10/393721 was filed with the patent office on 2003-10-23 for confocal microscope apparatus.
This patent application is currently assigned to Olympus Optical Co., Ltd.. Invention is credited to Nakata, Tatsuo.
Application Number | 20030197924 10/393721 |
Document ID | / |
Family ID | 29207521 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030197924 |
Kind Code |
A1 |
Nakata, Tatsuo |
October 23, 2003 |
Confocal microscope apparatus
Abstract
A confocal microscope apparatus comprises a first optical
scanning system which obtains a scan image of a sample using a
laser beam from a first laser light source, a second optical
scanning system which scans specific regions of a sample with a
laser beam from a second laser light source that is different from
the first laser light source, thereby causing a particular
phenomenon, and a beam diameter varying mechanism which can change
the beam diameter of the laser beam of at least one of the first
optical scanning system and the second optical scanning system.
With this configuration, the apparatus further comprises an
excitation light intensity distribution calculator which calculates
and stores the excitation light intensity distribution along a
depth direction on the sample surface from the beam diameter of the
laser beam output from the beam diameter varying mechanism.
Inventors: |
Nakata, Tatsuo; (Hino-shi,
JP) |
Correspondence
Address: |
FRISHAUF, HOLTZ, GOODMAN & CHICK, PC
767 THIRD AVENUE
25TH FLOOR
NEW YORK
NY
10017-2023
US
|
Assignee: |
Olympus Optical Co., Ltd.
Tokyo
JP
|
Family ID: |
29207521 |
Appl. No.: |
10/393721 |
Filed: |
March 21, 2003 |
Current U.S.
Class: |
359/368 |
Current CPC
Class: |
G02B 21/06 20130101;
G02B 21/16 20130101; G02B 21/0052 20130101; G02B 21/24 20130101;
G02B 21/008 20130101; G02B 21/0076 20130101; G02B 21/32 20130101;
G02B 21/0072 20130101 |
Class at
Publication: |
359/368 |
International
Class: |
G02B 021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2002 |
JP |
2002-089878 |
Claims
What is claimed is:
1. A confocal microscope apparatus comprising: a first optical
scanning system which obtains a scan image of a sample using a
laser beam from a first laser light source; a second optical
scanning system which scans specific regions of a sample with a
laser beam from a second laser light source that is different from
the first laser light source, thereby causing a particular
phenomenon; and a beam diameter varying mechanism which can change
the beam diameter of the laser beam of at least one of the first
optical scanning system and the second optical scanning system.
2. The confocal microscope apparatus according to claim 1, further
comprising an excitation light intensity distribution calculator
which calculates and stores the excitation light intensity
distribution along a depth direction on the sample surface from the
beam diameter of the laser beam output from the beam diameter
varying mechanism.
3. A confocal microscope apparatus comprising: a first optical
scanning system which scans a sample via an objective lens with
incoherent light output from an incoherent light source, and
detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of the sample with laser beam output from a laser
light source, thereby causing a particular phenomenon, wherein the
first optical scanning system further comprises a rotatable disk to
obtain a confocal effect, the light output from the incoherent
source scans the sample via the rotatable disk, and the
fluorescence is detected via the rotatable disk.
4. The confocal microscope apparatus according to claim 3, wherein
the second optical scanning system further comprises a beam
diameter varying mechanism which changes a beam diameter of the
laser beam of the laser light source.
5. The confocal microscope apparatus according to claim 4, further
comprising an excitation light intensity distribution calculator
which calculates and stores the excitation light intensity
distribution along the depth direction on the sample surface from
the beam diameter of the laser beam output from the beam diameter
varying mechanism.
6. The confocal microscope apparatus according to claim 3, wherein
the incoherent light source includes a lamp or an LED light
source.
7. A confocal microscope apparatus comprising: a first optical
system which illuminates a sample via an objective lens with
incoherent light output from an incoherent light source, and
detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of a sample with a laser beam from a laser light
source, thereby causing a particular phenomenon.
8. The confocal microscope apparatus according to claim 7, wherein
the second optical scanning system comprises a beam diameter
varying mechanism which changes the beam diameter of the laser beam
of the laser light source.
9. The confocal microscope apparatus according to claim 8, further
comprising an excitation light intensity distribution calculator
which calculates and stores the excitation light intensity
distribution along the depth direction on the sample surface from
the beam diameter of the laser beam output from the beam diameter
varying mechanism.
10. The confocal microscope apparatus according to claim 1, wherein
the first laser light source is an IR pulsed laser, and the beam
diameter varying mechanism is provided to the second scanning
optical system.
11. The confocal microscope apparatus according to claim 10,
further comprising a depth direction intensity distribution
calculator which calculates an intensity distribution along a depth
direction of the laser light beam output from the beam diameter
varying mechanism on the sample surface.
12. An observation method using a confocal microscope comprising:
irradiating an excitation light to a sample to excite the sample;
irradiating an light to cause the particular phenomenon to a
desired position; and imaging by detecting a light from the excited
sample, wherein said irradiating the excitation light includes
adjusting an intensity distribution of the excitation light along a
depth direction on the surface.
13. An observation method using a confocal microscope comprising:
irradiating an excitation light to a sample to excite the sample;
irradiating an light to cause the particular phenomenon to a
desired position; and imaging by detecting a light from the excited
sample, wherein said irradiating the sample includes adjusting an
intensity distribution of the light to cause the particular
phenomena along a depth direction on the surface.
14. An observation method using a confocal microscope comprising:
irradiating an excitation light to a sample via a ratatable disk to
acquire a fluorescent image of the sample by a disk scanning; and
irradiating an light to cause the particular phenomenon to a
desired position.
15. The observation method according to claim 14, wherein said
irradiating the light includes adjusting an intensity distribution
of the excitation light along a depth direction on the surface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is based upon and claims the benefit of
priority from the prior Japanese Patent Application No. 2002-89878,
filed Mar. 27, 2002, the entire contents of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a confocal microscope apparatus
which excites a specimen which has been marked with a fluorescent
dye or fluorescent protein using the excitation wavelength, and
detects fluorescence emitted from the specimen.
[0004] 2. Description of the Related Art
[0005] A scanning laser microscope has been proposed, which
includes a first optical scanning system for obtaining a scan image
of a sample and a second optical scanning system for causing a
particular phenomenon in specific areas on the sample surface
(refer to Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, the
entire contents of which are incorporated herein by reference). In
this laser scanning microscope, a specific area on the sample
surface is irradiated using a laser light source and an optical
path of the first optical scanning system, thus stimulating the
sample or a chemical substance injected into the sample. A specific
area on the sample surface which is different from the
above-mentioned area is excited using a laser light source and an
optical path of the second optical scanning system, and the
fluorescence is detected, and imaging is carried out. In the
specification, unless stated otherwise, an optical scanning system
for obtaining images of a sample is called a "first optical
scanning system" and an optical scanning system for causing a
particular phenomenon in specific areas of a sample is called a
"second optical scanning system".
[0006] Generally, in the confocal microscope, the focal point on
the sample surface and the conjugated focal point thereof are
provided before the detection device, and a pinhole is provided
therein. Thereby, the resolution of the sample along the depth
direction is 1.22 .lambda./NA, and a smaller confocal effect is
being utilized than when a regular microscope is used for
observation. There is resolution as a result of this confocal
effect, and thus a sharp cross sectional image (that is, an image
to obtain a thin slice image along depth direction) can be obtained
for the sample which is being scanned.
[0007] When the image is taken at a high speed or when a dark
sample is being used, the confocal effect is weakened by opening
the pinhole (that is, enlarging a diameter of the pinhole), and the
image is made bright by lowering the resolution of the
fluorescence.
[0008] Thus the confocal microscope has the pinhole and decreases
the resolution, and thus depth-direction information can be
obtained. However, since the focal depth of the sample is
determined by the flux diameter of the coherent light which is
irradiated on the objective lens, it is impossible to change the
focal depth at the pinhole.
[0009] Meanwhile, Koehler illumination is often used as the
lighting to the sample by the microscope. This Koehler illumination
along the thickness direction of the cross section of the sample
causes almost uniform excitation.
[0010] In the conventional confocal microscope described above,
when the apparatus is realized by using 2 laser scanning paths and
one objective lens, the excitation light intensity distribution
along the depth direction on the sample surface of the laser beam
for sample stimulation and the laser beam for obtaining images are
almost the same since only wavelength differences is generated.
BRIEF SUMMARY OF THE INVENTION
[0011] A confocal microscope apparatus according to a first aspect
of the present invention is characterized by comprising: a first
optical scanning system which obtains a scan image of a sample
using a laser beam from a first laser light source; a second
optical scanning system which scans specific regions of a sample
with a laser beam from a second laser light source that is
different from the first laser light source, thereby causing a
particular phenomenon; and a beam diameter varying mechanism which
can change the beam diameter of the laser beam of at least one of
the first optical scanning system and the second optical scanning
system.
[0012] A confocal microscope apparatus according to a second aspect
of the present invention is characterized by comprising: a first
optical scanning system which scans a sample via an objective lens
with incoherent light output from an incoherent light source, and
detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of the sample with laser beam output from a laser
light source, thereby causing a particular phenomenon, in which the
first optical scanning system further comprises a rotatable disk to
obtain a confocal effect, the light output from the incoherent
source scans the sample via the rotatable disk, and the
fluorescence is detected via the rotatable disk.
[0013] A confocal microscope apparatus according to a third aspect
of the present invention is characterized by comprising: a first
optical system which illuminates a sample via an objective lens
with incoherent light output from an incoherent light source, and
detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of a sample with a laser beam from a laser light
source, thereby causing a particular phenomenon.
[0014] Advantages of the invention will be set forth in the
description which follows, and in part will be obvious from the
description, or may be learned by practice of the invention.
Advantages of the invention may be realized and obtained by means
of the instrumentalities and combinations particularly pointed out
hereinafter.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0015] The accompanying drawings, which are incorporated in and
constitute a part of the specification, illustrate embodiments of
the invention, and together with the general description given
above and the detailed description of the embodiments given below,
serve to explain the principles of the invention.
[0016] FIG. 1 is a schematic diagram of a confocal microscope
apparatus according to a first embodiment of the invention;
[0017] FIG. 2 is a view showing a structural example of a first
beam diameter varying mechanism and a second bean diameter varying
mechanism;
[0018] FIG. 3 is a schematic diagram of a confocal microscope
apparatus according to a second embodiment of the invention;
[0019] FIG. 4 is a view showing an example of a rotatable disk used
in the invention; and
[0020] FIG. 5 is a view schematically showing a nerve tissue
observation.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Embodiments of the present invention will be described with
reference to the drawings.
[0022] (First Embodiment)
[0023] FIG. 1 is a schematic diagram of a confocal microscope
apparatus according to a first embodiment of the invention.
[0024] In FIG. 1, the confocal microscope apparatus comprises: a
first optical scanning system 100 for observation (or for obtaining
images) which scans a focal surface of a sample 134 with a laser
beam from a first laser light source 101; and a second optical
scanning system 200 for radiating the laser beam output from the
second laser light source 201 onto an optional position on the
sample 134, and splitting the caged reagent (i.e. for sample
stimulation). An optical path of the first optical scanning system
100 and an optical path of the second optical scanning system 200
meet at a dichroic mirror 120. As a result, the first optical
scanning system 100 and the second optical scanning system 200
share an objective lens 132.
[0025] In the first optical scanning system 100 and the second
optical scanning system 200, the coherent light output from the
first laser light source 101 arrives at the dichroic mirror 120 by
way of a first beam diameter varying mechanism 102 and a first
optical scanning unit 104. Also, the coherent light output from the
second laser light source 201 reaches the dichroic mirror 120 by
way of a beam diameter varying mechanism 202 and a second optical
scanning unit 203.
[0026] In addition, the first beam diameter varying mechanism 102
and the second beam diameter varying mechanism 202 are connected
electrically or indirectly to an excitation light intensity
calculator 160. As a result, the excitation light intensity
calculator 160 can obtain beam diameter information of the beams
output from the first beam diameter varying mechanism 102 and the
second beam diameter varying mechanism 202.
[0027] The first beam diameter varying mechanism 102 and the second
beam diameter varying mechanism 202 may, as shown in FIG. 2 for
example, include a plurality of mechanisms, which changes the flux
diameter such as beam expanders, on a rotatable turret. Also
mechanisms, in which optical elements such as a plurality of lenses
are combined, and the flux diameter is changed while the coherence
of the laser is maintained (for example, zoom mechanism), may be
adopted as the first beam diameter varying mechanism 102 and the
second beam diameter varying mechanism 202.
[0028] The operation of the confocal microscope apparatus according
to the first embodiment, which has the above-described
configuration, will be described.
[0029] The first optical scanning system 100 and the second optical
scanning system 200 are used for radiating a coherent light at an
optional (desired) position on the sample 134. Specifically, this
is as described below.
[0030] That is, the flux diameter of the coherent light generated
from the first laser light source 101 and the second laser light
source 201 respectively, are varied (adjusted) with the first beam
diameter varying mechanism 102 and the second beam diameter varying
mechanism 202.
[0031] The light beam output from the first beam diameter varying
mechanism 102 passes a dichroic mirror 150, and is arbitrarily
deflected to an XY direction by each of scanning mirrors 104a and
104b of the first optical scanning unit 104. The deflected light
beam is reflected at the mirror 106 after passing through a relay
lens 105, and then irradiated onto the dichroic mirror 120.
Meanwhile, the light beam output from the second beam diameter
varying mechanism 202 is suitably deflected in an XY direction by
each of scanning mirrors 203a and 203b the second optical scanning
unit 203. The deflected light beam passes through the relay lens
and is irradiated onto the dichroic mirror 120, and the optical
path is deflected at the dichroic mirror 120.
[0032] In addition, the coherent light from the dichroic mirror 120
is irradiated onto an image formation lens 130. By changing the
flux diameters of the laser beams from the first laser light source
and the second laser light source at the first beam diameter
varying mechanism 102 and the second beam diameter varying
mechanism 202 with respect to the pupil diameter of the objective
lens 132, the width of the excitation light distribution (and/or
the intensity distribution) along the depth direction on the
surface of the sample 134 corresponding to each of the optical
scanning systems can be changed.
[0033] The light beam that has passed through the image formation
lens 130 reaches the objective lens 132, passes through the
objective lens 132 and is focused on an arbitrary cross section 138
of the sample 134 which is mounted on a stage 136. The stage 136 is
movable along the XY horizontal direction and the height direction
(Z axis direction--the direction of the arrow in FIG. 1).
[0034] As described above, when the sample 134 is being scanned, in
accordance with the application, a particular field may be scanned
by each of the scanning mirrors 203a and 203b or it may be kept
still and irradiated in spots. Further by skipping each of the
scanning mirrors 203a and 203b momentarily, the field can be
irradiated in spots at a number of arbitrary positions from moment
to moment. Meanwhile, the coherent light generated from the first
laser light source 101 is transmitted by the dichroic mirror 150 as
described above, and it is deflected by each of the scanning
mirrors 104a and 104b of the first optical scanning unit 104.
[0035] When light beam is irradiated on the sample 134 by the first
optical scanning system 100, the fluorescent marker chemical is
excited and fluorescence is generated.
[0036] The fluorescence from the sample 134 takes the opposite
direction of the optical path of the light irradiated on the sample
134 and passes from the objective lens 132 by way of the image
formation lens 130, the dichroic mirror 120, the first optical
scanning unit 104, the relay lens 103, each of the scanning mirrors
104a and 104b and arrives at the dichroic mirror 150, and at the
dichroic mirror 150. The fluorescence is reflected and incident to
a photometry filter 140.
[0037] The light beam is incident to the photometry filter 140 and
only the fluorescent wavelength from the sample 134 is selected,
and the light beam from the sample 134 having only the fluorescence
wavelength is focused at a surface of the pinhole 144 by a lens
142. The fluorescence, which has passed through the pinhole 144, is
measured by a photoelectric conversion device 146.
[0038] The excitation light intensity calculator 160 calculates the
excitation light intensity distribution on the sample surface by
inputting the information on the beam diameter of the beam output
by the first beam diameter varying mechanism 102 and the second
beam diameter varying mechanism 202 and the performance
(specification) of the objective lens being used at the time. It
also has other functions such as outputting values, which have
already been stored in a memory, to interfaces such as a computer
or a display (not shown in the figure).
[0039] According to the confocal microscope apparatus of the first
embodiment of the invention as mentioned above, when the sample 134
is observed and recorded by the first optical scanning system 100,
by irradiating coherent light on the sample 134 by the second
optical scanning system 200, the dynamics (chemical reactions) of
sample 134 which are caused by the coherent light irradiation by
the second optical scanning system 200 can be adjusted by the first
optical scanning system 100.
[0040] In this case, in the first embodiment, the excitation light
distribution along the depth direction on the sample surface by the
first optical scanning system 100 and the second optical scanning
system 200 are independently set by the first beam diameter varying
mechanism 102 and the second beam diameter varying mechanism 202.
Accordingly, even if the excitation light distribution is narrow
for the field of the sample being excited by the second optical
scanning system, that is, even in the case where a large area of
the sample along the thickness direction is excited, by broadening
the excitation light distribution of the first optical scanning
system, it is possible to carry out observation.
[0041] Also, unlike the case described above, at the second optical
scanning system, a wide field of the sample along the thickness
direction is stimulated, and at the first optical scanning system,
the excitation light distribution field along the thickness
direction is narrowed, and thus the cross section 138 of the sample
can be observed with high resolution.
[0042] The first embodiment may be configured such that an IR pulse
laser is used as the first laser light source 101, and a
fluorescent image is obtained by two photon absorption. In this
case, the two photon absorption phenomenon occurs only at the
position where the image is formed and theoretically the pinhole 44
is unnecessary. Also, because the dichroic mirror 150 can transmit
the IR pulse laser, reflect the visible fluorescence and lead it to
the photoelectric converter 146, this embodiment has the property
of reflecting short wavelengths. It is also configured such that
the beam diameter varying mechanism 102 is not used.
[0043] As described above, by using an IR pulse laser as the first
laser light source 101, the configuration of the first optical
scanning system 100 is simplified. In addition, even in the case
where the first beam diameter varying mechanism 102 is not used,
the width of the excitation light distribution along the depth
direction on the sample surface of the optical scanning system 1
becomes narrow than the excitation light distribution along the
depth direction of the second optical scanning system 200 due to
the two photon absorption phenomenon. Further, in the case where
the thickness of the sample to be stimulated is to be changed, the
width of the excitation light distribution of the second optical
scanning system 200 can be made smaller by the second beam diameter
varying mechanism 202.
[0044] (Second embodiment)
[0045] A confocal microscope according to a second embodiment of
the invention is described with reference to FIG. 3. FIG. 3 is a
schematic diagram of the confocal microscope apparatus according to
the second embodiment of the invention. The second optical scanning
system 200 of FIG. 3 is the same as that of the first embodiment,
and has been assigned the same reference numbers and thus detailed
descriptions thereof are omitted.
[0046] In the second embodiment, a first optical scanning system
100' has a incoherent light source such as a mercury light source,
a halogen light source, or an LED light source as a light source
301. An optical lens 302, a polarizing plate 303 and a polarizing
beam splitter (PBS) 304 are arranged on an optical path of a light
beam emitted from the light source 301.
[0047] A rotatable disk 305, a first image formation lens 307, a
quarter wave plate 308, and objective lens 309 are arranged on a
reflection optical path of the PBS 304, and light beam from the
light source is incident to a sample 310 by way of these.
[0048] The rotatable disk 305 is connected to a shaft of a motor
(not shown) via a rotation shaft 306, and rotates at a
predetermined rotation speed. The rotatable disk 305 has linear
transmit portions through which light passes and linear shield
portions which shield light are alternately arranged. In addition,
the line width of the shield portion is wider than that of the
transmit portion, and for example, the ratio of the width of the
shield portion to that of the transmit portion is 1:9 (refer to
FIG. 4).
[0049] If the width of the portion through which light passes is W,
and as is the case with the pinhole, assuming that magnification
with which the specimen image is projected onto the disk is M, the
wavelength of the light is .lambda., and the numerical aperture of
the objective lens is NA,
W=k.lambda.M/NA
[0050] where k is a coefficient and a value in the range of 0.5 to
1 is often used for k.
[0051] Also, a CCD camera 312 is arranged on the transmission
optical path of the PBS 304 via a second image formation lens 311.
A monitor 313 for observing the image taken by the CCD camera 312
is connected to the camera 312.
[0052] The operation of the confocal microscope of the second
embodiment having the above configuration will be described in the
following.
[0053] The light beam output from the light source 301 passes
through the optical lens 302, and at the polarizing plate 303 it is
transformed to linearly polarized light having only a predetermined
polarization, and then input into the PBS 304. The PBS 304 reflects
the deflected light beam in the direction in which the beam has
passed through the polarizing plate and a light in a direction
parallel thereto is transmitted.
[0054] The light beam reflected at the PBS 304 is input into the
rotatable disk 305 which rotates at a predetermined speed. The
light beam passing through the transmit portion of the rotatable
disk 305 passes through the first image formation lens 307 and
becomes circularly polarized at the quarter wave plate 308, and is
focused with the objective lens 309 to be irradiated on the sample
310.
[0055] The light beam reflected by the sample 310 passes through
the objective lens 309, and at the quarter plate 308 it becomes
linearly polarized light which is orthogonal to that at the time of
input, thereby focusing the image of the sample 310 on the
rotatable disk 305, via the second imaging lens 311.
[0056] The focused component of the formed image formed on the
rotatable disk 305 passes through the transmit portion of the
rotatable disk 305. The component passing through the rotatable
disk 305 is transmitted by the PBS 304, and arrives at the CCD
camera 312 by way of the second image formation lens 311. The
specimen image is formed on the image formation surface (image
pickup surface).
[0057] If a particular moment when the sample 310 is being observed
is considered, a line is projected on the sample 310 along a
particular direction as shown in FIG. 4.
[0058] In this situation, in the case where the light beam
reflected from the sample 310 in this state is focused on the
rotatable disk 305, a line is projected on the rotatable disk 305
for the portion of the sample 310 which is in focus. However, for
the unfocused portion, the image that is projected on the rotatable
disk 305 is blurred, and thus most of the unfocused image cannot be
transmitted. Accordingly, only images which are in focus are
transmitted to the rotatable disk 305.
[0059] When the rotatable disk 305 is not rotating, the situation
is not changed and the image is simply one in which the sample and
the line overlap. However, by rotating the rotatable disk 305, the
line which includes the transmit portion and the shield portion
moves while changing its direction on the sample 310, and thus
there is uniformity, the line image disappears and an image which
is in focus can be observed. Thus, if the rotation of the rotatable
disk 305 is sufficiently fast with respect to the exposure time of
the CCD camera 312, the focused image can be picked up by the CCD
camera 312 and observed at the monitor 313. For example, if the CCD
camera 312 has a TV rate as a usual, the exposure time is {fraction
(1/60)} second or {fraction (1/30)} second. Therefore, the number
of rotations of the rotatable disk 305 during the exposure time
should be about 1800 rpm at which half revolutions occur.
[0060] The excitation light distribution along the depth direction
on the surface of the sample 310 of the first optical scanning
system 100' at this time is the same as the light distribution of
Koehler illumination of the microscope in the longitudinal
direction of the slit. At the width direction of the slit, the
distribution is the same as the second optical scanning system.
[0061] Accordingly, excitation light distribution along the depth
direction on the sample surface of the first optical scanning
system is a distribution of which both longitudinal direction and
width direction distributions are combined. It is possible to
change the excitation light intensity distribution along the depth
direction, by varying the width of the slit and the space between
the slits of the rotatable disk 305.
[0062] In the second embodiment, by detecting the dynamic change
which caused reaction of the light radiated by the second optical
scanning system which has been shown in the first embodiment using
the first optical scanning system 100', the excitation light
distribution along the depth direction on the surface of the first
and second samples can be different. Accordingly, a wider field of
measurement is possible in the first optical scanning system 100'
than the stimulation field in the second optical scanning system
200.
[0063] Particularly in nervous system measurements, in order to
catch movements of the nerve which extend along the thickness
direction of the sample, it is necessary to obtain the images with
high speed. Usually, with the confocal microscope apparatus, in
order to increase the width of the excitation light distribution
along the depth direction on the surface of the sample, if the
sample is extends along the thickness direction, the image cannot
be captured with one measurement. As a result, as in the second
embodiment, by reducing the width of the excitation light
distribution along the depth direction on the surface of the
sample, image measurements for wider fields can be taken.
Accordingly, the second embodiment may have a configuration in
which the rotatable disk 305 is omitted. Also the rotatable disk is
not limited to the structure shown in FIG. 4. Provided that the
confocal effect can be obtained, any configuration or structure can
be used. For example, the rotatable disk may be one having pinholes
formed therein, and it can be a reflection type rotatable disk
rather than the transmit type of the above-described
embodiment.
[0064] In addition, in the second embodiment, the second beam
diameter varying mechanism 202 is not necessarily needed. However,
if the second embodiment has the second beam diameter varying
mechanism 202, it is possible to change the proportion of the first
cross section and the second cross section, and by fine adjustment
of the field for obtaining images and the portion for stimulation,
the degree of freedom of the experiment (and/or observation) is
increased. In addition, when the second beam diameter varying
mechanism 202 is provided, it is preferable that the excitation
light intensity distribution calculator 60 is provided as in the
case of the first embodiment.
[0065] Also, in the above-described configuration, by the first
optical scanning system 100' having an optical microscope system
with Koehler illumination, it becomes possible for the image to be
obtained in a wider excitation field. In this case, the rotatable
disk 305 is unnecessary.
[0066] In the above-described second embodiment, the PBS 304 may be
replaced with a dichroic mirror. In this case, the light beam from
the light source is reflected at the dichroic mirror, and the
fluorescence from the sample passes through the dichroic mirror.
Thus the optical path of the optical excitation system and that of
the optical measurement system can be separated, and as a result
the polarizing plate 303 is unnecessary.
[0067] Applications of the confocal microscope apparatus of each of
the above-described embodiments include for example, the
application in the field of cell research in which the cell is
locally excited and reactions at the excited regions are
observed.
[0068] In the method known as the uncaged method, by locally
exciting the cell, the concentration of the activated material is
changed. When this change in concentration is to be measured, by
measuring peripheral portions other than the locally excited
regions simultaneously, the internal functions of the cell can be
analyzed.
[0069] In the photo-bleach method, by locally exciting the cell,
the excited regions are discolored. The phenomenon is seen where
due to migration of proteins from the periphery, over time color
returns to the region which has been discolored. Accordingly,
measurements for both the locally stimulated region and the
peripheral portions are necessary.
[0070] An example thereof is shown using FIG. 5. FIG. 5 is a
schematic view showing a nerve tissue observation.
[0071] For example, when ions transmitted on an axis cylinder 3
from a cell body 1 to a cell body 2 are observed with the caged
fluorescent dyes introduced into the cell body 1 as a probe, first
a laser beam for stimulating a sample is radiated on a focal point
surface 4 of the cell body 1. Next, subsequent changes are observed
with a laser beam for sample observation. However, the excitation
light intensity distribution of the laser beam for sample
observation along the depth direction usually has the same depth as
the excitation light intensity distribution 5 of the laser beam for
sample stimulation. Thus, in the prior art, the fluorescent dye
which transmits the axis cylinder 3 and is not within that
distribution field cannot be observed because it is not exposed to
excitation light. To the contrary, in each of the embodiments of
the invention, the excitation light intensity distribution along
the depth direction, of the laser beam for sample stimulation and
the laser beam for obtaining images on the surface of the sample
are each independently varied, thus solving the problem of the
prior art.
[0072] The inventions described in the following are extracted from
the embodiments described below. The above-described embodiments do
not limit the invention. Accordingly various modifications may be
made within the scope of the general inventive concept of the
invention.
[0073] The confocal microscope apparatus according to a first
aspect of the present invention is characterized by comprising: a
first optical scanning system which obtains a scan image of a
sample using a laser beam from a first laser light source; a second
optical scanning system which scans specific regions of a sample
with a laser beam from a second laser light source that is
different from the first laser light source, thereby causing a
particular phenomenon; and a beam diameter varying mechanism which
can change the beam diameter of the laser beam of at least one of
the first optical scanning system and the second optical scanning
system. By combining the optical laser system and the laser
scanning microscope, it becomes possible change the width of
measurement by using differences in the excitation intensity
distribution along the depth direction on the surface of the
sample. Specifically, this is done in the following manner.
[0074] Conventionally, when movement of the sample is being
analyzed, it is of course desirable for the field of excitation and
the field for obtaining the images to be different, and also for
the excitation light intensity distribution on the sample surface
of the laser beam for sample stimulation along the depth direction
and the excitation light intensity distribution on the sample
surface of the laser beam for obtaining images along the depth
direction to be different from each other. In addition, it is
desirable for the width of the excitation light intensity
distribution along the depth direction to be intentionally made
small.
[0075] In the first aspect, a beam diameter varying mechanism for
changing the beam diameter of the laser beam is provided to the
output exit for the laser beam of each of the optical scanning
systems. When the flux diameter is reduced by this beam diameter
varying mechanism, the numerical aperture of the objective lens is
smaller than in the case where the flux diameter is large.
Consequently, the width of the excitation light intensity
distribution along the depth direction on the surface of the sample
can be reduced without changing the objective lens. Further, by
providing each of the optical systems with the beam diameter
varying mechanism, the excitation light intensity distribution
along the depth direction of the sample surface of each of the
optical systems can be changed independently. Also, the excitation
light distribution along the depth direction on the sample surface
can be changed intentionally.
[0076] The confocal microscope apparatus according to a second
aspect of the present invention is characterized by comprising: a
first optical scanning system which scans a sample via an objective
lens with incoherent light output from an incoherent light source,
and detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of the sample with laser beam output from a laser
light source, thereby causing a particular phenomenon, in which the
first optical scanning system further comprises a rotatable disk to
obtain a confocal effect, the light output from the incoherent
source scans the sample via the rotatable disk, and the
fluorescence is detected via the rotatable disk. The optical laser
system and the disk type confocal microscope apparatus are
combined, so that it becomes possible to change the width for
measurement due to differences in the excitation intensity
distribution along the depth direction on the surface of the
sample.
[0077] The confocal microscope apparatus according to a third
aspect of the present invention is characterized by comprising: a
first optical system which illuminates a sample via an objective
lens with incoherent light output from an incoherent light source,
and detects fluorescence emitted from the sample via the objective
lens; and a second optical scanning system which irradiates
specific regions of a sample with a laser beam from a laser light
source, thereby causing a particular phenomenon. The optical laser
system and the microscope of Koehler illumination are combined, so
that it becomes possible to change the width of measurement due to
differences in the excitation intensity distribution along the
depth direction on the surface of the sample.
[0078] Preferred embodiments of the confocal microscope described
above are as described in the following. Each of the embodiments
may be used alone or may used in combination.
[0079] (1) The second optical scanning system further comprises a
beam diameter varying mechanism, which changes a beam diameter of
the laser beam of the laser light source.
[0080] (2) An excitation light intensity distribution calculator
which calculates and stores the excitation light intensity
distribution along a depth direction on the sample surface from the
beam diameter of the laser beam output from the beam diameter
varying mechanism is further provided.
[0081] (3) The first laser light source is an IR pulsed laser, and
the beam diameter varying mechanism is provided to the second
scanning optical system.
[0082] (4) In (3), a depth direction intensity distribution
calculator which calculates an intensity distribution along a depth
direction of the laser light beam output from the beam diameter
varying mechanism on the sample surface is further provided.
[0083] (5) The incoherent light source includes a lamp or an LED
light source.
[0084] The observation method according to the fourth aspect of the
invention is characterized by comprising: irradiating an excitation
light to a sample to excite the sample; irradiating an light to
cause the particular phenomenon to a desired position; and imaging
by detecting a light from the excited sample, in which said
irradiating the excitation light includes adjusting an intensity
distribution of the excitation light along a depth direction on the
surface.
[0085] The observation method according to the fifth aspect of the
invention is characterized by comprising: irradiating an excitation
light to a sample to excite the sample; irradiating an light to
cause the particular phenomenon to a desired position; and imaging
by detecting a light from the excited sample, in which said
irradiating the sample includes adjusting an intensity distribution
of the light to cause the particular phenomena along a depth
direction on the surface.
[0086] The observation method according to the sixth aspect of the
invention is characterized by comprising: irradiating an excitation
light to a sample via a ratatable disk to acquire a fluorescent
image of the sample by a disk scanning; and irradiating an light to
cause the particular phenomenon to a desired position. With this
configuration, it is preferable that the irradiating the light
includes adjusting an intensity distribution of the excitation
light along a depth direction on the surface.
[0087] According to the present invention, by independently
changing the intensity distribution along the depth direction on
the sample surface of the excitation light in the optical system
for sample excitation and for obtaining images, it becomes possible
to do dynamic analysis of different three-dimensional spaces.
[0088] Additional advantages and modifications will readily occur
to those skilled in the art. Therefore, the invention in its
broader aspects is not limited to the specific details and
representative embodiments shown and described herein. Accordingly,
various modifications may be made without departing from the spirit
or scope of the general inventive concept as defined by the
appended claims and their equivalents.
* * * * *